Methods for the generation of multispecific and multivalent antibodies

ABSTRACT

The invention provides novel bispecific monoclonal antibodies carrying a different specificity for each binding site of the immunoglobulin molecule and methods for producing novel bispecific monoclonal antibodies carrying a different specificity for each binding site of the immunoglobulin molecule. The antibodies are composed of a single heavy chain and two different light chains, one containing a Kappa constant domain and the other of a Lambda constant domain. The invention provides methods for the isolation of antibodies of different specificities but sharing a common heavy chain. The invention also provides methods for the controlled co-expression of two light chains and a single heavy chain leading to the assembly of monospecific and bispecific antibodies. The invention provides a mean of producing a fully human bispecific and bivalent antibody that is unaltered in sequence and does not involve the use of linkers or other non-human sequences, as well as antibody mixtures of two monospecific antibodies and one bispecific antibody. The invention also provides the means of efficiently purifying the bispecific antibody.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/050,815 filed on Oct. 10, 2013, which is a divisional of U.S.application Ser. No. 13/210,723, filed Aug. 16, 2011 and issued as U.S.Pat. No. 9,834,615, which claims the benefit of U.S. ProvisionalApplication No. 61/374,159, filed Aug. 16, 2010, U.S. ProvisionalApplication No. 61/443,008, filed Feb. 15, 2011, and U.S. ProvisionalApplication No. 61/509,260, filed Jul. 19, 2011, the contents of each ofwhich are hereby incorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “NOVI-023_D02US_SEQUENCELISTING.txt,” which was created on Mar. 29, 2012 and is 8.67 KB in size,are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the generation of novel bispecific monoclonalantibodies carrying a different specificity for each binding site of theimmunoglobulin molecule. The antibodies of the invention are composed ofa single heavy chain and two different light chains, one containing aKappa constant domain and the other of a Lambda constant domain. Thisinvention in particular relates to the isolation of antibodies ofdifferent specificities but sharing a common heavy chain. The inventionfurther relates to the controlled co-expression of two light chains anda single heavy chain leading to the assembly of monospecific andbispecific antibodies. The invention provides a mean of producing afully human bispecific and bivalent antibody that is unaltered insequence and does not involve the use of linkers or other non-humansequences, as well as antibody mixtures of two monospecific antibodiesand one bispecific antibody. The invention also provides the means ofefficiently purifying the bispecific antibody.

BACKGROUND OF THE INVENTION

An antibody is composed of four polypeptides: two heavy chains and twolight chains. The antigen binding portion of an antibody is formed bythe light chain variable domain (VL) and the heavy chain variable domain(VH). At one extremity of these domains six loops form the antigenbinding site and also referred to as the complementarity determiningregions (CDR). Three CDRs are located on the VH domain (H1, H2 and H3)and the three others are on the VL domain (L1, L2 and L3). During B celldevelopment a unique immunoglobulin region is formed by somaticrecombination known as V(D)J recombination. The variable region of theimmunoglobulin heavy or light chain is encoded by different genesegments. The heavy chain is encoded by three segments called variable(V), diversity (D) and joining (J) segments whereas the light chainvariable is formed by the recombination of only two segments V and J. Alarge number of antibody paratopes can be generated by recombinationbetween one of the multiple copies of the V, D and J segments that arepresent in the genome. The V segment encodes the CDR1 and CDR2 whereasthe CDR3 is generated by the recombination events. During the course ofthe immune response further diversity is introduced into the antigenbinding site by a process called somatic hypermutation (SHM). Duringthis process point mutations are introduced in the variable genes of theheavy and light chains and in particular into the regions encoding theCDRs. This additional variability allows for the selection and expansionof B cells expressing antibody variants with improved affinity for theircognate antigen.

The vast majority of immunoglobulins are bivalent and monospecificmolecules carrying the same specificity on both arms as they arecomposed of two identical heavy chain polypeptides and two identicallight chain polypeptides. However, it was recognized very early duringthe development of hybridoma technology that hybrid hybridomas can becreated by a fusion event between two hybridomas (Suresh M R et al.,Methods Enzymol 1986; 121: 210-228). These ‘quadromas’ express twodifferent heavy and two different light chains and therefore produce avariety of different antibody species resulting from the random pairingof the heavy and light chains. Amongst these different species,bispecific antibodies (bsAbs) are generated, carrying a differentspecificity on each arm. Another naturally occurring exception is theimmunoglobulin of the IgG4 isotype that is able to undergo heavy chainexchange due to a less stable dimerization mediated by the hinge regionof that isotype (van der Neut Kolfschoten M et al., Science. 2007317(5844):1554-7). Although this exchange seems to happen in vivo, itsbiological significance remains unclear.

Monoclonal antibodies have emerged as a successful and attractive classof molecules for therapeutic intervention in several areas of humandisease. However, targeting or neutralizing a single protein is notalways sufficient to achieve efficacy in certain diseases which limitsthe therapeutic use of monoclonal antibodies. It is increasingly clearthat in a number of indications neutralizing one component of abiological system is not sufficient to achieve efficacy. One solution tothis problem is the co-administration of several monoclonal antibodies.This approach is however complicated by regulatory aspects if theantibodies to be combined have not been previously approvedindividually. Moreover, combination approaches are also costly from amanufacturing perspective. Accordingly, there exists a need forantibodies and therapeutics that enable targeting of multiple antigenswith a single molecule.

SUMMARY OF THE INVENTION

The invention allows for the identification, production and purificationof bispecific antibodies that are undistinguishable in sequence fromstandard antibodies. The invention also allows for the production andpurification of a simple antibody mixture of three or more antibodiesall bearing the same heavy chain. The unmodified nature of theantibodies of the invention provides them with favorable manufacturingcharacteristics similar to standard monoclonal antibodies.

The bispecific antibodies of the invention are generated using thefollowing steps:

Two antibodies having different specificities and sharing the samevariable heavy chain domain but different variable light chain domainsare isolated. This step is facilitated by the use of antibody librarieshaving a fixed heavy chain or transgenic animals containing a single VHgene.

The variable heavy chain domain is fused to the constant region of aheavy chain, one light chain variable domain is fused to a Kappaconstant domain and the other variable light chain domain is fused to aLambda constant domain. Preferably, the light chain variable domainfused to the Kappa constant domain is of the Kappa type and the lightchain variable domain fused to the Lambda constant domain is of theLambda type. However the invention also enables the generation of hybridlight chains so that two variable light chain domains of the same typecan be used to generate bispecific antibodies of the invention.

The three chains are co-expressed in mammalian cells leading to theassembly and secretion in the supernatant of a mixture of threeantibodies: two monospecific antibodies and one bispecific antibodycarrying two different light chains. The ratio of the differentantibodies depends on the relative expression of the chains and theirassembly into an IgG. The invention provides methods to tune theseratios and maximize the production of bispecific antibody.

The antibody mixture is purified using standard chromatographytechniques used for antibody purification. The antibody mixture can becharacterized and used as a multi-targeting agent.

The bispecific antibody is purified using in a consecutive manneraffinity chromatography media that bind specifically to human Kappa andhuman Lambda constant regions. This purification process is independentof the sequence of the light chain variable domains and is thus genericfor all bispecific antibodies of the invention.

The isolated bispecific antibody bearing a light chain containing aKappa constant domain and a light chain containing a Lambda constantdomain is characterized using different biochemical and immunologicalmethods.

The bispecific antibody of the invention can be used for therapeuticintervention or as a research or diagnostic reagent.

The invention provides monoclonal antibodies carrying a differentspecificity in each combining site and including two copies of a singleheavy chain polypeptide and a first light chain and a second lightchain, wherein the first and second light chains are different.

In some antibodies, at least a first portion of the first light chain isof the Kappa type and at least a portion of the second light chain is ofthe Lambda type. In some antibodies, the first light chain includes atleast a Kappa constant region. In some antibodies, the first light chainfurther includes a Kappa variable region. In some antibodies, the firstlight chain further includes a Lambda variable region. In someantibodies, the second light chain includes at least a Lambda constantregion. In some antibodies, the second light chain further includes aLambda variable region. In some antibodies, the second light chainfurther includes a Kappa variable region. In some antibodies, the firstlight chain includes a Kappa constant region and a Kappa variableregion, and the second light chain includes a Lambda constant region anda Lambda variable region.

In some embodiments, the constant and variable framework regionsequences are human.

The invention also provides methods to produce and generate a bispecificantibody by a) isolating an antibody or antibody fragment region havinga specificity determined by a heavy chain variable domain combined witha first light chain variable domain; b) isolating an antibody orantibody fragment region having a different specificity determined bythe same heavy chain variable domain as the antibody of step a) combinedwith a second light chain variable domain; c) co-expressing in a cell:(i) the common heavy chain variable domain fused to an immunoglobulinheavy chain constant region; (ii) the first light chain variable domainfused either to a light chain constant domain of the Kappa type or fusedto a light chain constant domain of the Lambda type; and (iii) thesecond light chain variable domain fused to a light chain constantdomain of a different type than the first variable constant domain.

Some methods also include the additional step of d) isolating thebispecific antibodies produced from the monospecific antibodiesproduced. For example, in some methods, the isolation is accomplished byusing an affinity chromatography purification step. In some methods, thepurification step is performed using Kappa constant domain specific,Lambda constant domain or both Kappa constant domain specific and Lambdaconstant domain specific affinity chromatography media.

In some methods, a Kappa light chain variable domain is fused to aconstant region of the Kappa type. In some methods, a Kappa light chainvariable domain is fused to a constant region of the Lambda type. Insome methods, a Lambda light chain variable domain is fused to aconstant region of the Kappa type. In some methods, a Lambda light chainvariable domain is fused to a constant region of the Lambda type.

In some methods, step a) and b) are facilitated by the use of antibodylibraries having a common heavy chain and diversity confined to thelight chain variable domain. The variable heavy chain domain that isfoxed in one of such libraries can be based on different variablegermline genes and have different sequences both in the CDR andFramework regions. In some methods, such libraries were designed usingdifferent types of variable heavy chain domains and could be used togenerate antibodies of the invention.

In some methods, the antibody library is displayed on filamentousbacteriophage, at the surface of yeast, bacteria or mammalian cells orused for ribosome or other type of in vitro display.

The invention also provides methods of preparing a bispecific antibodythat specifically binds to a first antigen and a second antigen, whereinthe first and second antigens are different, by a) providing a firstnucleic acid molecule encoding a first polypeptide comprising a heavyvariable chain region of an immunoglobulin polypeptide or fragmentthereof that binds the first antigen coupled to an immunoglobulinconstant region; b) providing a second nucleic acid molecule encoding asecond polypeptide comprising a light chain variable region of theimmunoglobulin polypeptide or fragment thereof that binds the firstantigen coupled to a first Kappa-type or Lambda-type light chainconstant region; c) providing a third nucleic acid molecule encoding athird polypeptide comprising a light chain variable region of animmunoglobulin polypeptide or fragment thereof that binds the secondantigen coupled to a second Kappa-type or Lambda-type light chainconstant region, wherein the first and second light chain constantdomains are different types; and d) culturing a host cell comprising thefirst, second and third nucleic acid molecules under conditions thatpermit expression of the first, second and third polypeptides.

Some methods also include the further step of e) recovering thebispecific antibody. For example, in some methods, the bispecificantibody is recovered in step e) using an affinity chromatographypurification step. In some methods, the purification step is performedusing Kappa constant domain specific, Lambda constant domain or bothKappa constant domain specific and Lambda constant domain specificaffinity chromatography media.

In some methods, the second nucleic acid encodes a Kappa-type lightchain variable domain. In some methods, the second nucleic acid encodesa Kappa-type constant region. In some methods, the second nucleic acidencodes a Lambda-type constant region. In some methods, the secondnucleic acid encodes a Lambda-type light chain variable domain. In somemethods, the second nucleic acid encodes a Kappa-type constant region.In some methods, the second nucleic acid encodes a Lambda-type constantregion. In some methods, the third nucleic acid encodes a Kappa-typelight chain variable domain. In some methods, the third nucleic acidencodes a Kappa-type constant region. In some methods, the third nucleicacid encodes a Lambda-type constant region. In some methods, the thirdnucleic acid encodes a Lambda-type light chain variable domain. In somemethods, the third nucleic acid encodes a Kappa-type constant region. Insome methods, the third nucleic acid encodes a Lambda-type constantregion.

The invention also provides an antibody mixture that includes twomonospecific antibodies and one bispecific antibody, all having a commonheavy chain. For example, the bispecific antibody is any of thebispecific antibodies described herein or made using methods describedherein. The invention also provides methods of generating such anantibody mixture by a) isolating an antibody or antibody fragment regionhaving a specificity determined by a heavy chain variable domaincombined with a first light chain variable domain; b) isolating anantibody or antibody fragment region having a different specificitydetermined by the same heavy chain variable domain as the antibody ofstep a) combined with a second light chain variable domain; c)co-expressing in a cell: (i) the common heavy chain variable domainfused to an immunoglobulin heavy chain constant region; (ii) the firstlight chain variable domain fused either to a light chain constantdomain of the Kappa type or fused to a light chain constant domain ofthe Lambda type; and (iii) the second light chain variable domain fusedto either to a light chain constant domain of the Kappa type or fused toa light chain constant domain of the Lambda type. Some methods alsoinclude the additional step of d) isolating the antibody mixtureproduced in step c) from cell culture supernatant.

The invention also provides methods for two or more, for example, threeor more non-identical antibodies in a single recombinant host cell by a)expressing in the single recombinant host cell one or more nucleic acidsequences encoding a common immunoglobulin heavy chain and at least two,for example, at least three, different immunoglobulin light chains thatare capable of pairing with the common immunoglobulin heavy chain toform functional antigen binding domains to produce two or more, forexample, three or more, non-identical antibodies that comprise thecommon heavy chain. Some methods also include the step of harvesting orotherwise purifying the two or more, for example, three or more,non-identical antibodies from the recombinant host cell or from aculture of the host cell. The host cell is, for example, a mammaliancell. In some methods, non-identical antibodies include monospecific andbispecific antibodies.

In some methods, the non-identical antibodies target differing epitopesof the same target antigen. In some methods, the non-identicalantibodies have differing affinities for the same target epitope. Insome methods, the non-identical antibodies bind to different antigens.

In some methods, the two or more, for example, three or more,non-identical antibodies are independently selected from the groupconsisting of: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM.

In some methods, the two or more, for example, three or more,non-identical antibodies contain a modified Fc region that modifies theeffector functions of the antibodies such as Antigen Dependent Cellmediated Cytotoxicity (ADCC), Complement Dependent Cytotoxiciyt (CDC),Antigen Dependent Cellular Phagocytosis (ADCP) or their pharmacokineticproperties by altering its binding the neonatal Fc Receptors.

In some methods, the two or more, for example three or more differentimmunoglobulins are in the F(ab′)2 format.

In some methods, the one or more nucleic acid sequences are stablyexpressed in the host cell.

In some methods, the two or more, for example three or more,non-identical antibodies are produced by the host cell in vitro.

Some methods also include the additional steps of selecting at least onehost cell by assaying the two or more, for example, three or more,non-identical antibodies produced by the recombinant host cell for theirability to bind a target antigen; culturing the recombinant host cell;and isolating the three or more non-identical antibodies. The antibodiescan be isolated using any of the techniques described herein or anyother suitable art-recognized method.

In some methods, the different immunoglobulin light chains haveidentical constant regions. In some methods, the differentimmunoglobulin light chains have different constant regions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are a series of schematic representations of differentbispecific antibody formats. FIG. 1A depicts formats based on antibodyfragments: X-Link Fab, cross-linked Fab fragments; tascFv/BiTE,tandem-scFv/Bispecific T cell Engager; Db, diabody; taDb, tandemdiabody. FIG. 1B depicts formats based on Fc-fusions: Db-Fc, diabody-Fcfusion; taDb-Fc fusion, tandem diabody-Fc fusion; taDb-CH3, tandemdiabody-CH3 fusion; (scFv)₄-Fc, tetra scFv-Fc fusion; DVD-Ig, dualvariable domain immunoglobulin. FIG. 1C depicts IgG formats: knob-holeand SEED, strand exchange engineered domain; CrossMab, knob-holecombined with heavy and light chain domain exchange; bsAb, quadromaderived bispecific antibody; sdAb, single domain based antibody.

FIGS. 2A-2C are a series of schematic representations of possible modesof action enabled by bispecific antibodies. FIG. 2A depicts targeting oftwo antigens. FIG. 2B depicts retargeting of a toxic moiety or activityto a target cell. FIG. 2C depicts increased selectivity mediated byavidity.

FIGS. 3A-3C are a schematic representation of the structure of differentbispecific antibodies of the invention composed of two copies of aunique heavy chain polypeptide and two different light chainpolypeptides. The locations and/or arrangements of the Kappa light chainand the Lambda light chain (or portions thereof) shown in these figuresare not intended to be limiting. Those of ordinary skill in the art willappreciate that the Kappa light chain and the Lambda light chain (orportions thereof) can also be arranged so as to produce the mirror-imageof the bispecific antibodies shown in FIGS. 3A-3C. Those of ordinaryskill in the art will also appreciate that the bispecific antibodiesthat are represented in a full IgG format in FIGS. 3A-3C can also begenerated using other immunoglobulin isotypes or in other immunoglobulinformats such as F(ab′)2. 3A. Kappa variable domain fused to a Kappaconstant domain and Lambda variable domain fused to Lambda constantdomain. 3B. Kappa variable domains fused to a Kappa constant domain anda Lambda constant domain. 3 C Lambda variable domains fused to a Kappaconstant domain and a Lambda constant domain.

FIG. 4 is an illustration of an ELISA assay testing clones specific forhCXCL10-NusA or hIL6RC and bearing the same variable heavy chain domain.Each clone was tested against both targets to demonstrate specificity.

FIGS. 5A-C are a series illustrations depicting the three types oflibraries used in the Examples, for each library type, Vκ and Vλlibraries were kept separated. FIGS. 5A and C: Two sets of librariesthat contain a fixed VH3-23 variable domain that differ only by theirCDR H3 sequence that is indicated below the H3 (CDR definition accordingto IMGT). The light chain repertoire was diversified either by insertingrandomized sequences into the CDRL3 of selected light chain variablegenes (FIGS. 5A and 5C) or by capturing naturally rearranged light chainvariable domains isolated from human donors that can include all humanvariable genes and contain diversity in all 3 CDRs (FIG. 5B). Thedifferent diversification strategies are illustrated by horizontal linesbelow the diversified region of the light chain repertoires.

FIGS. 6A-6B are graphs depicting the results of ELISA using monospecificIgGλ and IgGκ selected against hIFNγ and IL6RC, respectively and bearinga common heavy chain. The ELISA formats are schematically representednext to each graph. In FIG. 6A, INFγ was immobilized on the plate,incubated with the anti-INFγ IgGλ or the anti-IL6RC (i.e., IL-6Rreceptor/IL-6 soluble complex) IgGκ and both were detected withanti-human Cκ or anti-human Cλ antibodies coupled to horse radishperoxidase. The signal was revealed by colorimetry and quantified usinga microtiter plate reader.

FIGS. 7A and 7B are a series of schematic representations of vectorsused for the co-expression of one heavy chain and two light chains inmammalian cells. Both vectors contain three promoters to drive geneexpression, a glutamine synthetase gene for stable cell line selection.In the second vector, pNovI κHλκ (FIG. 7B), the expression of anadditional Kappa light is driven by an internal ribosome entry site(IRES). The different genes and genetic control elements are indicated.hCMV, human cytomegalovirus promoter; SV40, V40 promoter; pApolyadenylation signal; VH, heavy chain variable domain; VK, light chainvariable Kappa domain; CK light chain constant Kappa domain; VL, lightchain variable Lambda domain; CL2, light chain constant Lambda domain2;GS cDNA, Glutamine Synthetase cDNA; AmpR;

-   -   selectable marker for Ampicilin resistance. A selected number of        restriction sites are indicated.

FIG. 8A is a schematic representation of the purification process forbispecific antibodies of the invention. FIG. 8B is an illustrationdepicting co-expression, purification and SDS-PAGE analysis ofbispecific antibodies of the invention. The gel was stained using simplyblue. PA: Protein A; K: Kappaselect; λ: Lambda selected; FT: columnflow-through; E: elution fraction.

FIG. 9 is an illustration of an SDS-PAGE analysis of total IgG purifiedfrom mammalian cells transfected with vectors enabling different levelsof Kappa light chain expression using different IRES elements within thepNovI κHλκ vector (lane 1-5) and compared to the pNovI κHλ vector. Therelative intensities of the Kappa and Lambda light chain indicate thatthe expression levels can be modulated.

FIG. 10A is an illustration depicting IEF gel analysis of purifiedmonospecific IgG (κκ and λλ) and bispecific IgG (κλ). FIG. 10B is anillustration of IEX-HPLC analysis of monospecific and bispecificantibodies. The three antibodies were injected independently and theirelution profile are overlaid in the graph. The gradient used in theexperiment is shown.

FIGS. 11A-11C are an illustration and series of graphs that depict theELISA assays used to determine the capacity of the bispecific antibodyto bind both target and the presence of a Kappa and a Lambda light chainin the molecule. FIG. 11A is a schematic representation of the ELISAformat. FIG. 11B is a graph depicting the results of the ELISA with INFγimmobilized on the plate. FIG. 11C is a graph depicting the results ofthe ELISA with IL-6RC immobilized on the plate. IgGκ, anti-IL6RCmonospecific antibody; IgGλ, anti-INFγ monospecific antibody; IgGκλ,anti-IL6RC/anti-INFγ bispecific antibody. Secondary detection antibodiesanti-human Lambda HRP and anti-Human Kappa HRP are indicated.

FIGS. 12A and 12B are a series of illustrations and graphs depicting SPRanalysis of IgGκλ bispecific antibodies. In FIG. 12A, INFγ wasimmobilized at the surface of the Biacore chip and anti-IL6RCmonospecific antibody (IgGκ), anti-INFγ monospecific antibody (IgGλ)and, anti-IL6RC/anti-INFγ bispecific antibody (IgGκλ) were injected onthe surface followed by injection of IL6RC. In FIG. 12B, theanti-IL6RC/anti-INFγ bispecific antibody (IgGκλ) was immobilized on thechip surface and anti human-Kappa and anti-human Lambda antibodies wereinjected at the same concentration. The experiment was repeatedinverting the order of antibody injection with identical results.

FIG. 13 is a schematic representation of an overview of one method ofgenerating the bispecific and multi-specific antibodies described hereinin CHO cells.

FIG. 14A is a graph depicting the growth and antibody productionprofiles of pools of CHO cells at a small-scale production level in anErlenmeyer flask. Antibody production levels were determined by ProteinA-HPLC analysis. VCC stands for viable cell concentration and Ab standsfor antibody. FIG. 14B is a graph depicting the growth and antibodyproduction profile comparison between small-scale and mid-scalefermentation. Antibody production levels were determined by ProteinA-HPLC analysis. VCC stands for viable cell concentration and Ab standsfor antibody.

FIGS. 15A and 15 B are a series of graphs depicting antibodyproductivity in a 96 well plate (96 wpl) of mono Kappa (KK), mono Lambda(LL) and bispecific Kappa Lambda (KL) antibody expressing cell linesfive weeks post-transfection in two independent experiments. Antibodyproduction levels were determined by ELISA. mAb stands for monoclonalantibody. FIGS. 15C and 15D are a series of graphs depicting antibodyproductivity in shaken 24 well plate (24 wpl) overgrown batch culturesof mono Kappa (KK), mono Lambda (LL) and bispecific Kappa.

FIG. 16A is an illustration depicting the results of reduced SDS-PAGEanalysis of monospecific κ IgG molecules (i.e., monospecific moleculeshaving Kappa light chains, also referred to herein as “mono κ”molecules), monospecific λ IgG molecules (i.e., monospecific moleculeshaving Lambda light chains, also referred to herein as “mono λ”molecules), and κλ antibodies (i.e., antibodies having both Kappa andLambda light chains) through the purification steps described above.FIG. 16B is an illustration depicting the results of reduced SDS-PAGEanalysis of mono κ, mono λ and κλ antibodies obtained following theelution steps described above. In FIGS. 16A and 16B, the gel was stainedusing simply blue, and E stands for elution fraction, FT stands forcolumn flow-through and MM stands for molecular weight marker. FIG. 16Cis an illustration depicting isoelectric focusing (IEF) gel analysis ofpurified monospecific IgG molecules (κκ and λλ) and the bispecific IgGmolecule (κλ).

FIGS. 17A-D are a series of graphs and illustrations depicting that themethods of generating bispecific antibodies of the invention produceantibodies that include both a Kappa light chain and a Lambda lightchain and that the purified antibodies exhibit bispecificity. The graphsdepict the results of ELISA using purified κλ-body against hIFNγ andIL6RC. The ELISA was performed using anti-Kappa or anti-Lambda detectionantibodies as indicated. FIGS. 17A-D illustrate that the Lambda lightchain binds to hIFNγ, while the Kappa light chain binds to IL6RC.NI-0501 is a control anti-hIFNγ Lambda light chain antibody, and NI-1201is a control anti-IL6RC Kappa light chain antibody.

FIG. 18 is an illustration of an IEF gel of different monospecific andbispecific antibodies, indicating that the difference in pI can varydepending on the antibody light variable sequence. Lane 1, anti-NusAIgGκ; Lane 2, anti-NusA/anti-INFγ IgGκλ; Lane 3, anti-INFγ IgGλ; Lane 4,anti-IL6RC IgGκ; Lane 5, anti-IL6RC/anti-IL6RC IgGκλ; Lane 6, anti-IL6RCIgGλ.

FIGS. 19A-19C are a series of schematic representations of threedifferent hybrid proteins obtained by combining a variable Lambda geneand a Kappa constant gene. The fusion points differ between thedifferent hybrids: in FIG. 19A, VLambda fused to CKappa; in FIG. 19B,VLambda up to CDR3 fused to VKappa FR4 and CKappa; and in FIG. 19C,VLambda and the first four amino acids of CLambda and CKappa excludingthe first four amino acids. CDR, Complementary Determining Region; FR,Framework region.

FIG. 20 is an illustration of the analysis of two hybrid light chainconstructs on a Bionalyzer 2100 system using a Protein 80 chip (AgilentTechnologies). The electropherogram corresponding to the gel image areindicated.

FIGS. 21A and 21B are a series of graphs depicting the results of doseresponse ELISA using scFv specific for INFγ (FIG. 21A) or IL6RC (FIG.21B) in which the VH domain was either the common VH originally selected(top curves) or other VH domains that allow scFv expression andpurification (bottom curves).

FIG. 22 is a graph depicting the results obtained for IgGκλ bispecificantibody quantification using a sandwich ELISA format. The dose responsewas performed using either purified bispecific antibody alone or mixedwith monospecific Kappa or Lambda antibodies at different ratios asindicated, in order to evaluate the interference of these molecules inthe assay.

DETAILED DESCRIPTION

In order to overcome the limitations of monoclonal and monovalentantibody therapeutics that can only target a single antigen or toovercome the limitations of combinations of monovalent antibodytherapeutics, intense efforts have aimed at multiple antigen targetingusing bispecific antibody formats. Such antibodies carrying more thanone specificity are of interest in biotechnology and have greatpotential as therapeutic agents enabling novel therapeutic approaches(Fischer and Leger, Pathobiology 2007; 74:3-14; Morrison S L NatureBiotechnol 2007; 25:1233-1234). Bispecific antibodies are advantageousas they allow for multiple targeting, they increase therapeuticpotential, they address redundancy of biological systems, and theyprovide novel mechanisms of action through abilities such as retargetingand/or increased specificity. As validated single therapeutic targetsbecome more and more exhausted, combinations allowed by bispecificantibodies provide a new and expansive universe of targets fortherapeutic agents and applications.

Several strategies have been used to generate such bispecific moleculessuch as chemical cross-linking of antibody fragments, forcedheterodimerization, quadroma technology, fusion of antibody fragmentsvia polypeptide linkers and use of single domain antibodies. Theavailability of recombinant DNA technologies has lead to the generationof a multitude of bispecific antibody formats (see e.g., Ridgway J B etal. (1996) Protein Eng 9: 617-621). Linkers and mutations havefrequently been introduced into different regions of the antibody toforce heterodimer formation or to connect different binding moietiesinto a single molecule. However, these engineered molecules often havepoor manufacturing characteristics, as well as an increased risk ofimmunogenicity, which limit or prevent their progression towards theclinic. In addition, prior attempts to develop bispecific formats havebeen limited due to factors such as poor stability and/or expression.These approaches are further described below and the formats discussedare illustrated in FIGS. 1A-1C.

Chemical Cross-Linking.

The use of chemical cross-linking reagents to covalently link twoantibodies is a conceptually straightforward approach. Antibodyfragments generated from their respective parent antibodies by enzymaticdigestion or generated through recombinant technologies are conjugatedusing bifunctional reagents (Glennie M J et al., J Exp Med 1992;175:217-225). Product homogeneity is the main limitation of thisapproach as the bispecific species has to be purified from homodimersand the modification steps can alter the integrity and stability of theproteins. The multiple steps involved make this approach challenging interms of manufacturing and product homogeneity.

Quadromas.

Quadromas and triomas can be generated by fusing either two hybridomasor one hybridoma with a B lymphocyte, respectively (Suresh M R et al.,Methods Enzymol 1986; 121: 210-228). In this case the simultaneousexpression of two heavy and two light chains leads to the randomassembly of 10 antibody combinations and the desired bsAb represent onlya small fraction of the secreted antibodies. The bsAb has to be purifiedusing a combination of chromatographic techniques, and dramaticallyreduces production yields. A major limitation is that quadromas producebsAb of rodent origin which limit their therapeutic potential due toimmunogenicity issues.

Recombinant Bispecific Antibodies.

The majority of bispecific antibody formats have been generated bygenetic engineering techniques using antibody fragment such as scFv orFab fragments as building blocks connected via polypeptide linkers.Formats based on linked antibody fragments include tandem scFv (BiTE),diabodies and tandem-diabodies (Kipriyanov S M. Methods Mol Biol 2003;207:323-333; Korn T et al., Int J Cancer 2002; 100:690-697). Thesebuilding blocks can further be linked to an immunoglobulin Fc regiongiven rise to ‘IgG-like’ molecules. These formats include diabody-Fc,tandem diabody-Fc, tandem diabody-CH3, (scFv)₄-Fc and DVD-Ig (Lu D etal., J Immunol Methods 2003; 279: 219-232; Lu D et al., J Biol Chem2005; 280: 19665-19672; Lu D et al., J Biol Chem 2004; 279: 2856-2865;Wu C et al., Nat Biotechnol 2007 25:1290-7). A potential limitation ofthe use of linkers is that the flexible nature of these peptides makesthem more prone to proteolytic cleavage, potentially leading to poorantibody stability, aggregation and increased immunogenicity. Inaddition, these foreign peptides might elicit an immune response againstthe junction between the protein and the linker or the linker itself. Ingeneral bsAbs based on linked building block are challenging moleculesin terms of manufacturing which limits their therapeutic potential.

An ideal bispecific molecule for human therapy should beundistinguishable from a normal IgG. Strategies based on forcing theheterodimerization of two heavy chains have been explored. A firstapproach coined ‘knob into hole’ aims at forcing the pairing of twodifferent IgG heavy chains by introducing mutations into the CH3 domainsto modify the contact interface (Ridgway J B et al., Protein Eng 1996;9: 617-621). On one chain amino acids with large side chains wereintroduced, to create a ‘knob’. Conversely, bulky amino acids werereplaced by amino acids with short side chains to create a ‘hole’ intothe other CH3 domain. By co-expressing these two heavy chains, more than90% heterodimer formation was observed (‘knob-hole’) versus homodimersformation (‘hole-hole’ or ‘knob-knob’). A similar concept was developedusing strand-exchange engineered domain (SEED) human CH3 domains basedon human IgG and human IgA sequences (Davis J H et al., 2010, PEDS23:195-202). These engineered domains lead to the formation ofheterodimeric molecules that can carry two different specificities.These two approaches are attractive as they favor the production of theheterodimer of interest (up to 95%) but do not fully prevent homodimerformation. Therefore downstream purification procedures capable ofremoving the homodimers from the heterodimers are still required.Another potential issue of these approaches is that the mutated domainsare not fully human and can lead to immunogenicity and might also affectthe domain stability and aggregation propensity of the molecule. Asthese strategies allow for the forced paring of the heavy chains, thedifferent light chains can randomly pair with any of the two heavychains and lead to the generation of different antibodies that need tobe purified from one another. Recently an improvement over the ‘knobinto hole’ approach has been described to solve the light chain pairingissue (WO 2009/080253 A1). This method involves the exchange of some ofthe light chain and heavy chain domains in addition to the ‘knob intohole’ mutations. The main advantage of this method is that a bispecificbivalent antibody having two different variable heavy chain domains andtwo different variable light chain domains can be generated and has beencoined “CrossMab.” However, the sequences of this bispecific antibodyare not fully human as it contains both mutations in the Fc to forceheterodimerization and non-natural junction points between the differentimmunoglobulin domains. Furthermore, these modifications lead to reducedexpression levels of the bispecific format compared to a standardmonoclonal antibody (Schaefer et al., PNAS 2011; 108:11187-11192).

Single Domain Based Antibodies.

The immune systems of camelids (lamas and camels) and cartilaginous fish(nurse sharks) use single V-domains fused to a Fc demonstrating that asingle domain can confer high affinity binding to an antigen. Camelid,shark and even human V domains represent alternatives to antibodies butthey also be used for bsAbs generation. They can be reformatted into aclassical IgG in which each arm has the potential to bind two targetseither via its VH or VL domain. This single domain-IgG would havebiochemical properties similar to an IgG and potentially solve problemsencountered with other bsAbs formats in terms of production andheterogeneity. It is however likely that steric hindrance will in oftenprevent simultaneous binding of both antigens on both antibody arms.

A representation of bispecific antibody formats described above is shownin FIGS. 1A-1C. Some of these format representations are derived fromFischer and Leger, Pathobiology 2007; 74:3-14; and Morrison S L NatureBiotechnol 2007; 25:1233-1234.

In contrast to these prior formats, the bispecific antibodies,multi-specific antibodies, compositions and methods provided hereinovercome such development obstacles. The bispecific antibodies providedherein have a common heavy chain, two light chains—one Kappa (κ), oneLambda (λ)—that each has a different specificity (i.e., two lightchains, two specificities). Preferably, the bispecific antibodies do notcontain any linkers or other modifications, including amino acidmutations. The methods provided herein produce molecules having specificbinding where diversity is restricted to the VL region. These methodsproduce the bispecific antibodies through controlled co-expression ofthe three chains (one VH chains, two VL chains), and purification of thebispecific antibody. The bispecific and/or multi-specific antibodiesdescribed herein exhibit similar affinities for a given target ascompared to the affinities of monospecific antibodies for that sametarget. Preferably, the bispecific and/or multi-specific antibodiesdescribed herein are virtually indistinguishable from standard IgGmolecules.

The methods provided herein also provide the means of generating simpleantibody mixtures of two monospecific antibodies and one bispecificantibody that are useful, for example, for multiple targeting withoutpurification of the bispecific antibody from the mixture.

Possible Modes of Action of Bispecific Antibodies

Simultaneous Inhibition of Two Targets.

By definition bispecific antibodies carry two specificities and cantherefore inhibit more than one target. These targets can be solublefactors or located on the surface of a cell. A number formats targetingmultiple cytokines have been generated successfully (Wu C et al., NatBiotechnol 2007 25:1290-7).

Retargeting.

As a majority of bispecific antibody formats are capable of binding twomolecules simultaneously, they can therefore be used as bridgingmolecules to retarget cytotoxic effector cells or cytotoxic agents tocells involved in a disease process. This application has been exploredin oncology. In some instances, one specificity of an antibody wasdirected against tumor cell markers such as CD19, CD20, HER2,carcinoembryonic antigen (CEA). The second arm of the bispecificantibody brings in close proximity a toxic moiety or activity such asdrugs, toxins, cytokines or an effector cell from the immune system (Tcells, NK cells, monocytes and neutrophils, by targeting CD3, CD16, CD64and CD89, respectively) (Thielemans K, Blood 1996; 87: 4390-4398;Goldstein J et al., J Immunol 1997; 158: 872-879).

Increased Specificity Via Avidity.

In a classical IgG format, antibody binding is directed both by theaffinity of each combining site for its antigen and on the avidityeffects provided by bivalent binding. The avidity effect dramaticallyincreases the apparent affinity of the antibody for cell surface markersas two dissociation events have to occur for the antibody to bereleased. Some of the bispecific formats described above are bivalent(i.e., one binding site for each target) whereas others are tetravalent.The latter have four binding sites or more and have the potential tobind each target in a bivalent manner. Bivalent bispecific astherapeutic agents selectively targeting cellular populations thatexpress a combination of cell surface markers. This unique mode ofaction is in principle restricted to molecules that can benefit from anavidity component to discriminate between cells expressing both antigensand those that express only one marker.

A representation of possible modes of action mediated by bispecificantibodies is shown in FIGS. 2A-2C. This representation is derived fromFischer and Leger, Pathobiology 2007; 74:3-14.

Characteristics and Limitations of Bispecific Antibody Formats

The key characteristics of current bispecific antibody formats aresummarized in Table I. All formats except those based on human domainscontain sequences that are not of human origin or contain non-humanprotein sequences generated by the fusion of different protein domains.The majority of formats using linkers lead to potential manufacturingissues due to domain aggregation. The presence of foreign sequences andunfavorable stability characteristics can potentially significantlyincrease the risk of immunogenicity. A key difference between formats isthe valency of their binding sites which is directly linked to theircapacity to mediate Retargeting or selective binding mediated byavidity. Thus all formats cannot enable all modes of action. Inparticular, the only format that is undistinguishable from a fully humanimmunoglobulin cannot mediate Retargeting or Increased Selectivityactivities. There is therefore a need to generate novel bispecificantibodies with favorable properties for the development oftherapeutics, i.e., being undistinguishable from a fully humanimmunoglobulin molecule, good manufacturability properties and enablingthe full spectrum of possible modes of actions.

TABLE I Characteristics of different bispecific antibody formats.Recombinant Cross- Recombinant Recombinant formats - based linkedformats - linked formats - forced on single fragments Quadromas antibodyfragments heterodimers domains Binding mode Bivalent BivalentTetravalent (or Bivalent Tetravalent more) Mode of action DI, R, IS DI,R, IS DI, IS DI, R, IS DI Manufacturing Complex, Purification Can bechallenging Simple mixture Simple multistep from a due to antibody(major part of the process complex fragment instability product ismixture of and aggregation bispecific) antibodies Sequence origin Human,Rodent Human, presence of Human, presence Human modified sequenceslinkers and non- of mutations to sites by the human protein force cross-junctions heterodimerization linking process Modes of action: DI, DualInhibition; R, Retargeting; IS, Increased Selectivity.

Improved Methods for Generating Bispecific and Bivalent Antibodies.

The present invention provides methods of generating bispecificantibodies that are identical in structure to a human immunoglobulin.This type of molecule is composed of two copies of a unique heavy chainpolypeptide, a first light chain variable region fused to a constantKappa domain and second light chain variable region fused to a constantLambda domain. Each combining site displays a different antigenspecificity to which both the heavy and light chain contribute. Thelight chain variable regions can be of the Lambda or Kappa family andare preferably fused to a Lambda and Kappa constant domains,respectively. This is preferred in order to avoid the generation ofnon-natural polypeptide junctions. However it is also possible to obtainbispecific antibodies of the invention by fusing a Kappa light chainvariable domain to a constant Lambda domain for a first specificity andfusing a Lambda light chain variable domain to a constant Kappa domainfor the second specificity (FIGS. 3A-3B). The bispecific antibodiesdescribed herein are also referred to as IgGκλ antibodies or “κλbodies,” a new fully human bispecific IgG format. This κλ-body formatallows the affinity purification of a bispecific antibody that isundistinguishable from a standard IgG molecule with characteristics thatare undistinguishable from a standard monoclonal antibody and,therefore, favorable as compared to previous formats.

An essential step of the method is the identification of two antibody Fvregions (each composed by a variable light chain and variable heavychain domain) having different antigen specificities that share the sameheavy chain variable domain. Numerous methods have been described forthe generation of monoclonal antibodies and fragments thereof. (See,e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporatedherein by reference). Fully human antibodies are antibody molecules inwhich the sequence of both the light chain and the heavy chain,including the CDRs 1 and 2, arise from human genes. The CDR3 region canbe of human origin or designed by synthetic means. Such antibodies aretermed “human antibodies”, or “fully human antibodies” herein. Humanmonoclonal antibodies can be prepared by using the trioma technique; thehuman B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today4: 72); and the EBV hybridoma technique to produce human monoclonalantibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies maybe utilized and may be produced by using human hybridomas (see Cote, etal., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforminghuman B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

Monoclonal antibodies are generated, e.g., by immunizing an animal witha target antigen or an immunogenic fragment, derivative or variantthereof. Alternatively, the animal is immunized with cells transfectedwith a vector containing a nucleic acid molecule encoding the targetantigen, such that the target antigen is expressed and associated withthe surface of the transfected cells. A variety of techniques arewell-known in the art for producing xenogenic non-human animals. Forexample, see U.S. Pat. Nos. 6,075,181 and 6,150,584, which is herebyincorporated by reference in its entirety.

Alternatively, the antibodies are obtained by screening a library thatcontains antibody or antigen binding domain sequences for binding to thetarget antigen. This library is prepared, e.g., in bacteriophage asprotein or peptide fusions to a bacteriophage coat protein that isexpressed on the surface of assembled phage particles and the encodingDNA sequences contained within the phage particles (i.e., “phagedisplayed library”).

Hybridomas resulting from myeloma/B cell fusions are then screened forreactivity to the target antigen. Monoclonal antibodies are prepared,for example, using hybridoma methods, such as those described by Kohlerand Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse,hamster, or other appropriate host animal, is typically immunized withan immunizing agent to elicit lymphocytes that produce or are capable ofproducing antibodies that will specifically bind to the immunizingagent. Alternatively, the lymphocytes can be immunized in vitro.

Although not strictly impossible, the serendipitous identification ofdifferent antibodies having the same heavy chain variable domain butdirected against different antigens is highly unlikely. Indeed, in mostcases the heavy chain contributes largely to the antigen binding surfaceand is also the most variable in sequence. In particular the CDR3 on theheavy chain is the most diverse CDR in sequence, length and structure.Thus, two antibodies specific for different antigens will almostinvariably carry different heavy chain variable domains.

The method of the invention overcomes this limitation and greatlyfacilitates the isolation of antibodies having the same heavy chainvariable domain by the use of antibody libraries in which the heavychain variable domain is the same for all the library members and thusthe diversity is confined to the light chain variable domain. Suchlibraries are described, for example, in co-pending applicationPCT/US2010/035619, filed May 20, 2010 and published on Nov. 25, 2010 asPCT Publication No. WO 2010/135558 and co-pending applicationPCT/US2010/057780, filed Nov. 23, 2010 each of which is herebyincorporated by reference in its entirety. However, as the light chainvariable domain is expressed in conjunction with the heavy variabledomain, both domains can contribute to antigen binding. To furtherfacilitate the process, antibody libraries containing the same heavychain variable domain and either a diversity of Lambda variable lightchains or Kappa variable light chains can be used in parallel for invitro selection of antibodies against different antigens. This approachenables the identification of two antibodies having a common heavy chainbut one carrying a Lambda light chain variable domain and the other aKappa light chain variable domain that can be used as building blocksfor the generation of a bispecific antibody in the full immunoglobulinformat of the invention. The bispecific antibodies of the invention canbe of different Isotypes and their Fc portion can be modified in orderto alter the bind properties to different Fc receptors and in this waymodifiy the effectors functions of the antibody as well as itpharmacokinetic properties. Numerous methods for the modification of theFc portion have been described and are applicable to antibodies of theinvention. (see for example Strohl, W R Curr Opin Biotechnol 2009(6):685-91; U.S. Pat. No. 6,528,624; PCT/US2009/0191199 filed Jan. 9,2009). The methods of the invention can also be used to generatebispecific antibodies and antibody mixtures in a F(ab′)2 format thatlacks the Fc portion.

Another key step of the invention is the optimization of co-expressionof the common heavy chain and two different light chains into a singlecell to allow for the assembly of a bispecific antibody of theinvention. If all the polypeptides get expressed at the same level andget assembled equally well to form an immunoglobulin molecule then theratio of monospecific (same light chains) and bispecific (two differentlight chains) should be 50%. However, it is likely that different lightchains are expressed at different levels and/or do not assemble with thesame efficiency. Therefore the methods of the invention also providemeans to modulate the relative expression of the different polypeptidesto compensate for their intrinsic expression characteristics ordifferent propensities to assemble with the common heavy chain. Thismodulation can be achieved via promoter strength, the use of internalribosome entry sites (IRES) featuring different efficiencies or othertypes of regulatory elements that can act at transcriptional ortranslational levels as well as acting on mRNA stability. Differentpromoters of different strength could include CMV (Immediate-earlyCytomegalovirus virus promoter); EF1-1α (Human elongation factor1α-subunit promoter); Ubc (Human ubiquitin C promoter); SV40 (Simianvirus 40 promoter). Different IRES have also been described frommammalian and viral origin. (See e.g., Hellen C U and Sarnow P. GenesDev 2001 15: 1593-612). These IRES can greatly differ in their lengthand ribosome recruiting efficiency. Furthermore, it is possible tofurther tune the activity by introducing multiple copies of an IRES(Stephen et al. 2000 Proc Natl Acad Sci USA 97: 1536-1541). Themodulation of the expression can also be achieved by multiple sequentialtransfections of cells to increase the copy number of individual genesexpressing one or the other light chain and thus modify their relativeexpressions. The Examples provided herein demonstrate that controllingthe relative expression of the different chains is critical formaximizing the assembly and overall yield of the bispecific antibody.

The co-expression of the heavy chain and two light chains generates amixture of three different antibodies into the cell culture supernatant:two monospecific bivalent antibodies and one bispecific bivalentantibody. The latter has to be purified from the mixture to obtain themolecule of interest. The method described herein greatly facilitatesthis purification procedure by the use of affinity chromatography mediathat specifically interact with the Kappa or Lambda light chain constantdomains such as the CaptureSelect Fab Kappa and CaptureSelect Fab Lambdaaffinity matrices (BAC BV, Holland). This multi-step affinitychromatography purification approach is efficient and generallyapplicable to antibodies of the invention. This is in sharp contrast tospecific purification methods that have to be developed and optimizedfor each bispecific antibodies derived from quadromas or other celllines expressing antibody mixtures. Indeed, if the biochemicalcharacteristics of the different antibodies in the mixtures are similar,their separation using standard chromatography technique such as ionexchange chromatography can be challenging or not possible at all.

The invention also provides a new means of producing simple antibodymixtures of two or more monospecific antibodies and one or morebispecific antibody that share the same heavy chain and can be purifiedusing standard chromatography techniques used for monoclonal antibodypurification. (See e.g., Lowy, I et al. N Engl J Med 2010; 362:197-205;Goudsmit, J. et al. J Infect Dis. 2006. 193, 796-801). Such simplemixtures can be used as multi-targeting agents for therapeutic usage.

Successful co-expression, purification and characterization of the heavychain and two light chains and purification of the bispecific antibodiesare shown in the Examples. The genes encoding the common heavy chain andthe two light chains were cloned into a vector containing threepromoters. After transient transfection, the supernatant of PEAK cellswas collected.

The co-expression of the three chains led to the assembly of threedifferent antibodies: two monospecific and one bispecific antibodies.Their theoretical relative ratios should be 1:1:2 provided theexpression levels and assembly rates are similar for both light chains.The bispecific antibodies were purified using a three-step affinitychromatography procedure: (1) Protein A: capture IgG (mono- and bi-),(2) Kappa select: capture IgG containing a Kappa light chain(s), and (3)Lambda select: capture IgG containing a Lambda light chain. Kappaselectand Lambdaselect are affinity chromatography media developed by BAC, BVand GE Healthcare.

The purified bispecific antibodies were characterized as follows. Theflow-through and elution from each affinity purification step wasanalyzed by SDS-PAGE. The results indicate that, at each step,bispecific antibodies are enriched (FIGS. 8A-8B). The κλ-body containedequivalent amounts of Kappa and Lambda light chains. The κλ-bodyexhibited an intermediate migration pattern on an isoelectric focusinggel and ion exchange chromatography compared to the two monospecificantibodies (FIGS. 10A-10B). The specificity and affinity of κλ-bodieswas determined by ELISA and surface plasmon resonance. The methods ofthe invention allow for the identification of antibodies with affinitiesin the sub-nanomolar to nanomolar range without optimization. This isnot obvious as the diversity in antibody libraries described herein isrestricted to the light chain which contributes less to the bindingenergy in standard antibodies.

To avoid the requirement of having access to two antibodies having lightchain variable domains of the Kappa and Lambda type being perceived as alimitation to the instant invention, the methods described herein allowfor the generation of hybrid light chain in which a Lambda variabledomain can be fused to a Kappa constant domain and conversely a Kappavariable domain can be fused to a Lambda constant domain as depicted inFIGS. 3B-3C. This widens the applications of the invention to antibodypairs that share a light chain of the same type. As described in theExamples provided herein, the fusion point between the variable andconstant domains is important and can affect the bispecific antibodypurification process.

An overview of one method of producing the bispecific and/ormulti-specific antibodies of the invention is shown in FIG. 13. In someembodiments, the methods of generating bispecific and/or multi-specificantibodies use a complete serum-free chemically defined process. Thesemethods incorporate the most widely used mammalian cell line inpharmaceutical industry, the Chinese Hamster Ovary (CHO) cell line. Themethods described therein are used to generate both semi-stable andstable cell lines. The methods can be used to manufacture the bispecificand/or multi-specific antibodies of the invention at small scale (e.g.,in an Erlenmeyer flask) and at mid-scale (e.g., in 25 L Wave bag). Themethods are also readily adaptable for larger scale production of thebispecific and/or multi-specific antibodies, as well as antibodymixtures of the invention.

The methods of generating the bispecific and/or multi-specificantibodies of the invention are advantageous because they employ genericpurification processes as shown in FIG. 8A. FIGS. 16A-16C demonstratepurification and product integrity testing of bispecific antibodiespurified from a semi-stable cell line. The bispecific antibodies werepurified using the following three-step affinity chromatographyprocedure: (i) Protein A purification to capture IgG molecules,including both monospecific and bispecific; (ii) KappaSelectpurification to capture IgG containing Kappa light chain(s); (iii)LambdaSelect purification to capture IgG containing Lambda light chain.The flow-through and elution from each affinity purification steps wereanalyzed by SDS-PAGE. The results demonstrated the removal of eachmonospecific form (i.e., monospecific IgG molecules having Kappa lightchains and monospecific IgG molecules having Lambda light chains) duringthe purification process (FIG. 16A). The purified κλ-containingantibodies (i.e., antibodies having both Kappa and Lambda light chains)contained equivalent amount of Kappa and Lambda light chains (FIG. 16B).The purified κλ-containing antibodies presented an intermediatemigration pattern on an isoelectric focusing gel as compared to the twomonospecific antibodies (FIG. 16C).

The chemically defined processes for manufacturing the bispecific and/ormulti-specific antibodies of the invention can be used with either poolsof CHO cells or with established cell lines. The results obtained withthe chemically defined process using either pools or established celllines demonstrate comparable productivities and growth characteristicsto those expressing the corresponding Kappa or Lambda monospecificantibodies. Thus, the κλ-body conserves both the structure andmanufacturing characteristics of a classical human IgG.

Previous approaches to produce bispecific antibody formats aimed atforcing the production of a homogenous bispecific molecule using thedifferent antibody engineering approaches described above were done atthe expense of productivity, scalability and stability of the product.The present invention is a different approach that allows the productionof a simple mixture of antibodies that have the standard characteristicsof productivity and scalability of monoclonal antibodies and providesefficient and generic means to purify the bispecific antibody from themixture or to purify the antibody mixture.

EXAMPLES Example 1: Generation of Antibody Libraries Having Fixed HeavyChains

Antibody libraries in which the heavy variable domain is identical forall the library members were generated as follows. First, the heavychain variable VH3-23 domain containing a defined CDR3 AKSYGAFDY (SEQ IDNO: 1) (CDR nomenclature according to IMGT) and a defined FR4 sequencewas cloned into the pNDS vector using the SfiI and XhoI restrictionsites to obtain the phagemid vector pNDS_VHfixed. The amino acidsequence of VK FR4 corresponds to the FR4 region encoded by the germlineJ genes JK1. The amino acid sequence of Vλ FR4 corresponds to the FR4region encoded by the germline J genes JL2 and JL3. Two variants of theVk FR4 sequence were generated with a single amino acid substitution atposition 106 (Arginine or Glycine). A total of 6 Kappa (VK1-33, VK1-39,VK3-11, VK3-15, VK3-20, VK4-1) and 5 Lambda variable domain genes(Vλ1-44, Vλ1-51, Vλ6-57, Vλ2-14, Vλ1-40) containing a stuffer fragmentinstead of a CDR3 encoding sequence were cloned into the pNDS_VHfixed inorder to generate 17 acceptor vectors in which diversity of synthetic ornatural origin can be cloned and high-diversity libraries can begenerated according to the methods described in co-pending applicationPCT/US2010/035619, filed May 20, 2010, published as WO2010/135558, andthe methods described in Ravn et al. (2010) Nucl Acid Res 38(21):e193,each of which is hereby incorporated by reference in its entirety. Thisprocess resulted in the generation of 17 libraries all having a commonVH3-23 domain and VKappa or VLambda domains diversified in the variablelight chain complementarity determining region 3 (CDRL3 region)containing a total of 6.9×10⁹ variants (FIG. 5A). Sequencing of 180randomly picked transformants indicated that >90% of the clones hadintegrated a CDRL3 sequence that was in-frame and therefore potentiallyfunctional.

Example 2: Phage Rescue of the Libraries

Each library was rescued independently according to standard phagedisplay procedures briefly summarized hereafter. A volume of cells fromthe frozen library aliquots sufficient to cover at least 10 times thetheoretical diversity of the library was added to 500 ml of 2×TYAG (100μg/ml ampicilin; 2% glucose) and grown at 37° C. with agitation (240rpm) until an OD600 of 0.3 to 0.5 was reached. The culture was thensuper-infected with MK13KO7 helper phage and incubated for one hour at37° C. (150 rpm). The medium was then changed by centrifuging the cellsat 2000 rpm for 10 minutes, removing the medium and resuspending thepellet in 500 ml of 2×TY-AK (100 μg/ml ampicilin; 50 μg/ml kanamycin).The culture was then grown overnight at 30° C. (240 rpm). The culturewas centrifuged at 4000 rpm for 20 minutes to pellet the cells. Thesupernatant was collected and 30% (vol/vol) of PEG 8000 (20%)/2.5M NaClwas added to precipitate the phage particles by incubating the mixture 1hour on ice. The phage particles were collected by centrifugation at10,000 rpm for 30 minutes and resuspended in 10 ml of TE buffer (10 mMtris-HCl pH 8.0; 1 mM EDTA). The resuspended solution was centrifuged at10,000 rpm to clear the bacterial debris and the precipitation procedurewas repeated. After final resuspension, phage was titrated by infectionof E. coli and absorption at 260 nm. The display level of scFv at thesurface of phage was also evaluated by Western blot analysis using ananti-c-myc monoclonal antibody. Purified phage from different librarieswas stored frozen at −80° C. after addition of glycerol to a finalconcentration of 15% (w/v).

Example 3: Phage Display Selections Using Fixed Heavy Chain Libraries

Liquid Phase Selections Against Biotinylated hCXCL10-NusA Fusion Protein(hCXCL10-NusA) and Biotinylated hIL6 Receptor Complex (hIL6RC):

Aliquots of the VH-Vκ and VH-Vλ phage libraries (10¹¹-10¹² Pfu) werekept separated and blocked with PBS containing 3% (w/v) skimmed milk forone hour at room temperature on a rotary mixer. Blocked phage was thendeselected on streptavidin magnetic beads (Dynal M-280) for one hour atroom temperature on a rotary mixer. Deselected phage was then incubatedwith in vivo biotinylated hCXCL10-NusA or hIL6RC (100 nM) for two hoursat room temperature on a rotary mixer. Beads were added to the targetand were captured using a magnetic stand followed by four washes withPBS/0.1% Tween 20 and 3 washes with PBS. Beads were then directly addedto 10 ml of exponentially growing TG1 cells and incubated for one hourat 37° C. with slow shaking (100 rpm). An aliquot of the infected TG1was serial diluted to titer the selection output. The remaining infectedTG1 were spun at 3000 rpm for 15 minutes and re-suspended in 0.5 ml2×TYAG (2×TY media containing 100 μg/ml ampicilin and 2% glucose) andspread on 2×TYAG agar Bioassay plates. After overnight incubation at 30°C., 10 ml of 2×TYAG was added to the plates and the cells were scrapedfrom the surface and transferred to a 50 ml polypropylene tube. 2×TYAGcontaining 50% glycerol was added to the cell suspension to obtain afinal concentration of 17% glycerol. Aliquots of the selection roundwere kept at −80° C.

Phage Rescue:

100 μl of cell suspension obtained from previous selection rounds wereadded to 20 ml of 2×TYAG and grown at 37° C. with agitation (240 rpm)until an OD600 of 0.3 to 0.5 was reached. The culture was thensuper-infected with 3.3×10¹⁰ MK13KO7 helper phage and incubated for onehour at 37° C. (150 rpm). The medium was then changed by centrifugingthe cells at 3800 rpm for 10 minutes, removing the medium andresuspending the pellet in 20 ml of 2×TY-AK (100 μg/ml ampicilin; 50μg/ml kanamycin). The culture was then grown overnight at 30° C. (240rpm). The next day an aliquot of the centrifuged supernatant was used asan input for the next round of selection.

Monoclonal Phage Rescue for ELISA:

Single clones were picked into a microtiter plate containing 150 μl of2×TYAG media (2% glucose) per well and grown at 37° C. (100-120 rpm) for5-6 h. M13KO7 helper phage was added to each well to obtain amultiplicity of infection (MOI) of 10 (i.e., 10 phage for each cell inthe culture) and incubated at 37° C. (100 rpm) for 1 h. Followinggrowth, plates were centrifuged at 3,200 rpm for 10 min. Supernatant wascarefully removed, cells resuspended in 150 μl 2×TYAK medium and grownovernight at 30° C. (120 rpm). For the ELISA, the phage are blocked byadding 150 μl of 2× concentration PBS containing 5% skimmed milk powderfollowed by one hour incubation at room temperature. The plates werethen centrifuged 10 minutes at 3000 rpm and the phage containingsupernatant used for the ELISA.

Phage ELISA:

ELISA plates (Maxisorp, NUNC) were coated overnight with 2 μg/mlhCXCL10-NusA in PBS or 2 μg/ml hIL6RC in PBS. Plates were then blockedwith 3% skimmed milk/PBS at room temperature for 1 h. Plates were washed3 times with PBS 0.05% Tween 20 before transferring the pre-blockedphage supernatants and incubation for one hour at room temperature. Eachclone was tested against both targets to test its specificity. Plateswere then washed 3 times with PBS 0.05% Tween 20. 50 μl of 3% skimmedmilk/PBS containing (HRP)-conjugated anti-M13 antibody (Amersham,diluted 1:10,000) to each well. Following incubation at room temperaturefor 1 hr, the plates were washed 5 times with PBS 0.05% Tween 20. TheELISA was then revealed by adding 50 μl of TMB (Sigma) and 50 μl of 2NH₂SO₄ to stop the reaction. Absorption intensity was read at 450 nm.Clones specific for hCXCL10-NusA or hIL6RC bearing the same variableheavy chain domain could be identified. (FIG. 4).

Phage Clone Sequencing:

Single clones were grown in 5 ml of 2×TYAG media (2% glucose) per welland grown at 37° C. (120 rpm) overnight. The next day phagemid DNA waspurified and used for DNA sequencing using a primer specific for pNDS1:mycseq, 5′-CTCTTCTGAGATGAGTTTTTG. (SEQ ID NO: 1).

Large Scale scFv Purification:

A starter culture of 1 ml of 2×TYAG was inoculated with a single colonyfrom a freshly streaked 2×TYAG agar plate and incubated with shaking(240 rpm) at 37° C. for 5 hours. 0.9 ml of this culture was used toinoculate a 400 ml culture of the same media and was grown overnight at30° C. with vigorous shaking (300 rpm). The next day the culture wasinduced by adding 400 μl of 1M IPTG and incubation was continued for anadditional 3 hours. The cells were collected by centrifugation at 5,000rpm for 10 minutes at 4° C. Pelleted cells were resuspended in 10 ml ofice-cold TES buffer complemented with protease inhibitors as describedabove. Osmotic shock was achieved by adding 15 ml of 1:5 diluted TESbuffer and incubation for 1 hour on ice. Cells were centrifuged at10,000 rpm for 20 minutes at 4° C. to pellet cell debris. Thesupernatant was carefully transferred to a fresh tube. Imidazole wasadded to the supernatant to a final concentration of 10 mM. 1 ml ofNi-NTA resin (Qiagen), equilibrated in PBS was added to each tube andincubated on a rotary mixer at 4° C. (20 rpm) for 1 hour. The tubes werecentrifuged at 2,000 rpm for 5 minutes and the supernatant carefullyremoved. The pelleted resin was resuspended in 10 ml of cold (4° C.)Wash buffer 1 (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH to 8.0).The suspension was added to a polyprep column (Biorad). 8 ml of coldWash Buffer 2 (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH to 8.0)were used to wash the column by gravity flow. The scFv were eluted fromthe column with 2 ml of Elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250mM imidazole, pH to 8.0). Fractions were analyzed by absorption at 280nm and protein containing fractions were pooled before buffer exchangeon a NAPS desalting column (Amersham) equilibrated with PBS. The scFv inPBS were analyzed by SDS-PAGE and quantified by absorption at 280 nm.The purified scFv were aliquoted and stored at −20° C. and at 4° C. Thepurified scFv were used in ELISA to confirm specific binding to thetarget against which they had been selected.

Example 4: Additional Selections Using Different Fixed VH Libraries

Antibody libraries were also generated by capturing naturally rearrangedlight chain repertoires and cloning them in the context of a single VHdomain described in Example 1. In this case the whole light chainvariable gene region was amplified from human cDNA using primers thatcorrespond to the 5′ and 3′ region of human rearranged variables regionsand cloned into pNDS_VHfixed vector described in Example 1. Another setof libraries was generated as described in Example 1 but using a fixedVH3-23 domain containing a different CDRH3 sequence ARGDDVS (SEQ IDNO:3). The libraries described above are schematically represented inFIGS. 5A-5C. These fixed VH libraries were used against a panel oftarget proteins using the selection and screening methodology describedin Example 2 and 3. Selections have been performed using the followingtargets: hCXCL10-NusA, IL-6RC, CD47, CD16, CD8 and hIFNγ. Candidatesthat were identified and shown to be specific for their targets arelisted in Table II. These results demonstrate that antibodies bindingdifferent targets and having a common heavy chain can be generated andthat diversity restricted to the light chain is sufficient to conferantigen specificity. Candidates could be generated from librariescontaining a VH3-23 domain with different CDRH3 sequences or having VLrepertoires diversified using different strategies. Therefore, theresults demonstrate that the approach is not restricted to a particularVH sequence or to a particular light chain variable domaindiversification strategy.

TABLE II Number of independent clones identified against a panel oftargets using libraries with a fixed VH sequence containing twodifferent CDRH3 sequences. Fixed CDRH3 Fixed CDRH3 Total SEQ ID: 1 SEQID: 3 Targets: κ λ κ λ κ λ hCD16 5 1 4 — — 1 hCD8 5 12 2 10 3 2 hCD47 219 16 4 5 5 IL6RC 17 14 8 7 9 7 IFNγ — 5 — 5 — — NusA-CXCL10 2 4 2 4 NANA NA: selection not performed.

Example 5: Fixed VH Candidates Reformatting into IgG and TransientExpression in Mammalian Cells

After screening, scFv candidates were reformatted into IgG and expressedby transient transfection into PEAK cells. Several IgGλ (n=5) and IgGκ(n=9) having a common heavy chain and specific for IFNγ and IL6RC,respectively, and having different light chain sequences were generatedas follow. The VH and VL sequences of selected scFv were amplified withspecific oligonucleotides and cloned into an expression vectorcontaining the heavy and light chain constant regions and theconstructions were verified by sequencing. The expression vectors weretransfected into mammalian cells using the Fugene 6 Transfection Reagent(Roche, Basel, Switzerland). Briefly, Peak cells were cultured in 6-wellplates at a concentration of 6×10⁵ cells per well in 2 ml culture mediacontaining fetal bovine serum. The expression vectors encoding thecandidate VH and VL sequences were co-transfected into the cells usingthe Fugene 6 Transfection Reagent according to manufacturer'sinstructions. One day following transfection, the culture media wasaspirated, and 3 ml of fresh serum-free media was added to cells andcultured for three days at 37° C. Following three days culture period,the supernatant was harvested for IgG purified on protein G-Sepharose 4Bfast flow columns (Sigma, St. Louis, Mo.) according to manufacturer'sinstructions. Briefly, supernatants from transfected cells wereincubated overnight at 4° C. with ImmunoPure (G) IgG binding buffer(Pierce, Rockford Ill.). Samples were then passed over ProteinG-Sepharose 4B fast flow columns and the IgG consequently purified usingelution buffer. The eluted IgG fraction was then dialyzed against PBSand the IgG content quantified by absorption at 280 nm. Purity and IgGintegrity were verified by SDS-PAGE. The IgG were then tested forbinding by to IFNγ and the IL-6/IL-6R receptor complex, referred toherein as IL6RC, by ELISA. Biotinylated IFNγ and IL6RC were immobilizedon streptavidin coated microplates (Streptawell, Roche) and the plateswere then blocked with PBS supplemented with 2% BSA at room temperaturefor 1 h. Plates were washed 3 times with PBS 0.05% Tween 20 beforeadding the purified anti-INFγ IgGλ or the anti-IL6RC IgGκ on wellscoated with either target. After one hour incubation at room temperatureand washing of the plates bound antibodies were detected with anti-humanCκ or anti-human Cλ antibodies coupled to horse radish peroxidase. TheELISA was then revealed by adding 50 μl of TMB (Sigma) and 50 μl of 2NH₂SO₄ to stop the reaction. Absorption intensity was read at 450 nm. Theresults indicate that the binding specificity of the scFv isolated fromthe fixed VH libraries was maintained in the IgG format and demonstratedthat two IgGs having the same heavy chain but different light chains canbe specific for distinct targets (FIGS. 6A-6B).

The heavy and light chain amino acid sequences of the anti-INFγ IgGλ orthe anti-IL6RC IgGκ are indicated below:

Anti-IL6RC VKappa light chain (SEQ ID NO: 4)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWLPTTPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECAnti-INTγ VLambda light chain (SEQ ID NO: 5)NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSQSWDGNHIVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS  Common heavy chain (SEQ ID NO: 6)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG.

Example 6: Co-Expression of a Single Heavy Chain and Two Light Chains inMammalian Cells

The simultaneous expression of one heavy chain and two lights chain inthe same cell can lead to the assembly of three different antibodies(FIG. 8A). Simultaneous expression can be achieved in different wayssuch as that the transfection of multiple vectors expressing one of thechains to be co-expressed or by using vectors that drive multiple geneexpression. A vector pNovi κHλ was designed to allow for theco-expression of one heavy chain, one Kappa light chain and one Lambdalight chain (FIG. 7A). The expression of the three genes is driven byhuman cytomegalovirus promoters (hCMV) and the vector also contains aglutamine synthetase gene (GS) that enables the selection andestablishment of stable cell lines. Another vector, pNovi κHλκ, was alsoconstructed, in which a second copy of a Kappa light chain was insertedafter the Lambda light chain and its expression was driven by aninternal ribosome entry site (IRES). Using this bicistronic design, therelative expression of the Kappa light chain can be increased. The twovectors are schematically represented in FIGS. 7A-7B.

Example 7: Transient Co-Expression of a Single Heavy Chain and Two LightChains in Mammalian Cells and Purification of Total IgG

The VH and VL gene of the anti-INFγ IgGλ or the anti-IL6RC IgGκ werecloned in the vector pNovi κHλ described in Example 5, for transientexpression in mammalian cells. Peak cells were cultured in 6-well platesat a concentration of 6×10⁵ cells per well in 2 ml culture mediacontaining fetal bovine serum. 2 μg of plasmid DNA was transfected intothe cells using TransIT-LT1 transfection reagent (Minis) according tomanufacturer's instructions. One day following transfection, the culturemedia was aspirated, and 3 ml of fresh serum-free media was added tocells and cultured for five days at 37° C. Following a five days cultureperiod, the supernatant was harvested, centrifuged at 4000 rpm andapplied on Mab Select Sure PA resin (GE Healthcare) according tomanufacturer's instructions. Total IgGs were eluted under acidiccondition followed by neutralization and buffer exchange into PBS. TheIgG content was quantified by absorption at 280 nm and analyzed bySDS-PAGE. The presence of two bands corresponding to the two lightchains and one band corresponding to the heavy chain indicated that thethree chains could be expressed simultaneously in the same cell (FIG.8B).

Example 8: Purification of Bispecific Antibodies Carrying a Lambda and aKappa Light Chain

The co-expression of one heavy chain and two light chains in the samecells can lead to the assembly of three different antibodies: twomonospecific antibodies bearing the same light chain on both arms and abispecific antibody bearing a different light chain associated with theheavy chain on each arm. The later can be isolated from the monospecificantibodies by chromatography techniques that take advantage of thedifferent biochemical properties of the monospecific and bispecificantibodies. In order to develop a generic purification approach that isapplicable to all bispecific antibodies generation, we used an affinitychromatography approach schematically depicted in FIG. 8A. Culturesupernatants from transfected cells were applied to Mab Select Sure PAresin (GE Healthcare) column and total IgG purified as described inExample 7. The total IgG was then applied to a column containingCaptureSelect Fab Kappa affinity matrix (BAC BV, Holland) equilibratedwith ten volumes of PBS. The column was then washed with 5-10 columnvolumes of PBS. The Immunoglobulin molecules bearing a Kappa light chainwere eluted from the column by applying 5 column volumes 0.1 M GlycinepH 2.0 and fractions were collected and neutralized. The fractionscontaining the antibody were pooled before buffer exchange on a PD10desalting column (Amersham) equilibrated with PBS. The antibody was thenapplied on a second column containing CaptureSelect Fab Lambda affinitymatrix equilibrated with ten volumes of PBS. The column was then washedwith 5-10 column volumes of PBS. The Immunoglobulin molecules bearingonly Kappa light chain do bind to the column and were found in theflowthrough. Antibodies carrying a Lambda light chain were eluted fromthe column by applying 5 column volumes 0.1 M Glycine pH 3.0 andfraction were collected. The fractions containing the bispecificantibody were pooled before buffer exchange on a PD10 desalting column(Amersham) equilibrated with PBS. The Immunoglobulin molecules bearing aKappa light chain were eluted from the column by applying 5 columnvolumes 0.1 M Glycine pH 2.0 and fractions were collected andneutralized. The fractions containing the antibody were pooled beforebuffer exchange on a PD10 desalting column (Amersham) equilibrated withPBS. The antibody was then applied on a second column containingCaptureSelect Fab Lambda affinity matrix equilibrated with ten volumesof PBS. The column was then washed with 5-10 column volumes of PBS. TheImmunoglobulin molecules bearing only Kappa light chain do bind to thecolumn and were found in the flowthrough. Antibodies carrying a Lambdalight chain were eluted from the column by applying 5 column volumes 0.1M Glycine pH 3.0 and fraction were collected. The fractions containingthe bispecific antibody were pooled before buffer exchange on a PD10desalting column (Amersham) equilibrated with PBS. The flow through andelution fraction of each purification step was analyzed by SDS-PAGE andindicated that the antibodies bearing Lambda light chains were found inthe flow through of the CaptureSelect Fab Kappa affinity matrix and,conversely, that antibodies bearing Kappa light chains were found in theflow through of the CaptureSelect Fab Lambda affinity matrix (FIG. 8B).As expected, in the final elution fraction, the intensity of the bandscorresponding to the two light chains is equivalent. Thus this threestep approach allows for the purification of bispecific antibodiesbearing both a Kappa and a Lambda light chain. The final recovery ofbispecific antibody bearing a Kappa and Lambda light chain wasapproximately 10-21% in different experiments. The Protein A elutionfraction also indicated that more Lambda light chain that Kappa lightchain is assembled into the purified IgG suggesting that increasedexpression of Kappa light chain could increase the assembly and recoveryof bispecific antibody.

Example 9: Modulation of Light Chain Expression to Optimize BispecificAntibody Assembly

In order to increase the expression of the Kappa light chain, the commonVH gene, the VLambda gene of the anti-INFγ antibody and two copies ofVKappa of the anti-IL6RC were cloned in the vector pNovi κHλ κ describedin Example 6. The expression of the second copy of the VKappa lightchain is driven by and IRES. Different IRES elements were tested toachieve different levels of VKappa light chain expression. This resultedin the construction of five independent vectors: pNovi κHλκ 1 to 5.These vectors were used for transient transfections in mammalian cellsas described in Example 7 and total IgG were purified form thesupernatant using Protein A affinity purification. The SDS-PAGE analysisindicates that the different pNovi κHλκ vectors increased the expressionand assembly of the Kappa light chain into the IgG compared toexpression using the pNovi κHλ vector (FIG. 9). The total IgG fractionsobtained after Protein A purification for each of these constructs wasfurther purified in two consecutive steps using CaptureSelect Fab Kappaand CaptureSelect Fab Lambda as described in Example 8. The yield inbispecific antibodies was 20% for the pNovi κHλ and ranged between 33and 41% for pNovi κHλκ constructs, indicating that increased Kappa lightchain expression lead to increased bispecific antibody assembly. Tofurther confirm these findings, co-transfections suing pNovi κHλ and thevector expressing the monospecific anti-IL6RC IgGκ were performed. Inthis way the relative levels of Kappa light chain as well as the yieldsof bispecific antibody were also increased indicating that severalapproaches can be taken to adjust the relative light chain expression.

Example 10: Characterization of the Purified Bispecific Antibody

The purified bispecific antibodies isolated as described in the Examplesabove were characterized using different techniques.

Isoelectric Focusing Gel (IEF).

The purified bispecific antibodies isolated as described in Example 9were analyzed using an IsoGel Agarose IEF plates with a range of pH 3-10(Lonza) and compared to the monospecific anti-INFγ IgGλ and theanti-IL6RC IgGκ antibodies. After focusing, the gel was placed infixative solution for 30 mins, washed with water for 5 min and thendried at RT. Gel was stained with Coomassie staining for 15 min, brieflyrinsed with water and with destaining solution for 2×15 min. Finally gelwas dried at RT before imaging. The results shown in FIG. 10A indicatethat the IgGκλ bispecific antibody has a different and intermediate pIcompared to the pI of the two monospecific antibodies as predicted fromthe antibody format and theoretical calculations. The staining alsodemonstrated that the bispecific antibody is highly pure after the threestep purification process described in Example 8.

Ion Exchange Chromatography (IEX).

50 μg of purified monospecific and bispecific antibodies were analyzedby Ion Exchange-High Performance Liquid Chromatography (IEX-HPLC) (HPLCWaters e2695/detector 2489) using an Agilent column Bio Mab, NP5,(Agilent): the mobile phases were: A: Na2HPO4/NaH2PO4 10 mM, pH6.5; B:Na2HPO4/NaH2PO4 10 mM, NaCl 100 mM, pH6.5; using a flow of 0.8 mL/minand 20%-60% gradient over 133 minutes. The three antibodies havedifferent retention times with the peak corresponding of the bispecificantibody having an intermediate profile compared to the monospecificantibodies (FIG. 10B).

ELISA.

The monospecific anti-INFγ IgGλ, the anti-IL6RC IgGκ and the bispecificIgGκλ antibodies were tested by ELISA for binding to INFγ and IL6RC. Theresults shown in FIGS. 11A-11C indicate that the bispecific IgGκλ isable to bind to both target and is can be detected with both anti-humanCκ and anti-human Cλ secondary antibodies.

Surface Plasmon Resonance (SPR).

The affinity and binding kinetics of monospecific anti-INFγ IgGλ, theanti-IL6RC IgGκ and the bispecific IgGκλ antibodies were characterizedon a Biacore 2000 instrument (Biacore AB, Uppsala, Sweden). 200 RU of agoat anti-human polyclonal IgG (ahIgG; Biacore) were immobilized byEDC/NHS chemistry on a CM5 Biacore chip. This surface was used tocapture monospecific or bispecific human IgG. The surface wasregenerated after each cycle by injection of 10 mM glycine pH=1.5 at 30μL/min, for 30 s followed by 1 min of stabilization time in HBS-EPbuffer. The data was fitted according to 1:1 Langmuir model and theK_(on), K_(off) and K_(D) values determined (Table III). Similaraffinity values were obtained for the monospecific antibodies and thebispecific IgGκλ antibodies. The data indicates that the two antibodycombining sites bind to hIFNγ and IL6RC similarly in a monospecific andbispecific format.

The capacity of the bispecific IgGκλ to interact with both targetssimultaneously was tested by SPR. Biotinylated INFγ was immobilized onstreptavidin coating CM5 Biacore chip. The monospecific and thebispecific antibodies were injected on this surface followed byinjection of IL6RC. The sensorgram shown in FIG. 11A show that thebispecific IgGκλ antibody was able to bind to immobilized INFγ and wasable to capture IL6RC simultaneously. SPR was also used to assess therelative amounts of Kappa and Lambda light chains in the purifiedbispecific antibody. IgGκλ was directly immobilized via amine couplingon the surface of a CM5 Biacore chip and an anti-human CKappa antibodywas injected followed by an anti-human CLambda antibody at the sameconcentration. Equivalent responses were obtained with both anti-lightchain antibodies indicating that, as predicted by the format, equivalentamounts of Kappa and Lambda light chains are present in the bispecificIgGκλ antibody (FIG. 12B)

TABLE III Binding kinetic analysis for monospecific and IgGκλ bispecificantibodies for IFNγ and IL6RC measured on a Biacore 2000 system. AnalyteLigand KD (M) k_(on) (1/Ms) k_(off) (1/s) IFNγ IgCκλ 1.84E−10 9.19E+051.69E−04 IgGλλ 1.96E−10 6.08E+05 1.19E−04 IL6RC IgGκλ 2.72E−07 7.44E+032.02E−03 IgGκκ 2.66E−07 8.08E+03 2.15E−03

Example 11: Manufacturing of Bispecific IgGκλ Antibodies

The expression of IgGκλ bispecific antibody was also performed inChinese Hamster Ovary (CHO) cells that are widely used for themanufacturing of monoclonal antibodies. In the example presented hereinboth semi-stable pools of transfected CHO cells as well as stable cellCHO lines were generated for the production of IgGκλ bispecificantibodies. In the studies presented herein, stable CHO lines weregenerated and grown using a chemically defined, animal component-free(CDACF) manufacturing process. The overall process is depicted in FIG.13.

Cho Pools.

CHO cells were electroporated with the linearized vector pNovi κHλκencoding the IgGκλ anti-INFγ/anti-IL6RC bispecific antibody described inthe Examples above as well as with plasmids driving the expression ofthe monospecific anti-INFγ IgGλ and the anti-IL6RC IgGκ. Afterelectroporation, pools of transfected cells were grown in non-fed 10 dayovergrown conditions. The cells transfected with bispecific constructpresented similar growth profiles as compared to the cells transfectedwith monospecific expression vectors. In addition, the productivitieswere also comparable and reached a typical range of antibodyproductivity: between 100-200 mg/L for non-fed overgrown pool cultures(FIG. 14A). Scaling up between small-scale production in an Erlenmeyerflask (100 mL) and mid-scale production in a 25 L Wave Bag wassuccessfully achieved (FIG. 14B). These results indicate that similargrowth curves and productivities are obtained during expression ofbispecific IgGκλ antibody in CHO cell lines and the correspondingmonospecific antibodies.

Stable CHO Cell Lines.

Recombinant cell lines producing bispecific antibody were generated byelectroporation of CHO cells with the pNovi κHλκ vector. Posttransfection, recombinant cell lines were selected by diluting the cellculture in the presence of a final concentration of 50 μM methioninesulphoximine (MSX). After 6 weeks of incubation, colonies of recombinantcell lines were screened for total IgG productivity by FastELISA® (R&DBiotech) (FIG. 15A-B). Selected cell lines were expanded in cell culturemedium containing 25 μM MSX, transferred to 24 wells microtitre platesand screened for productivity and growth characteristics in suspensionculture (FIG. 15C-D). The results revealed that the IgGκλ bispecificantibody can be produced in shaken batch overgrown conditions at a levelcomparable to cell lines expressing standard monospecific antibodies.Top producing cell lines were selected and operated in 50 mL batchovergrown cultures in shake flasks for a maximum of 10 days. Protein AHPLC was used for total IgG quantification in the supernatant. Total IgGfrom the supernatants of the 10 top producing cell lines were purifiedby MabSelect SuRE chromatography using 1 mL HiTrap (GE Healthcare)prepacked columns. The relative amounts of monospecific and bispecificantibodies in the total purified IgG was assessed by IEX-HPLC asdescribed in Example 10. For the majority of the cell lines, thefraction of IgGκλ bispecific antibody varied between 37-42% of the totalIgG and two cell lines had expressed lesser amounts of IgGκλ (22 and25%). The results for the 10 CHO cells lines are summarized in Table IV.

TABLE IV Total antibody Bispecific post antibody Total Total MabSelectrelative bispecific antibody SuRr quantity from antibody titrepurification IEX-HPLC quantity (mg/mL) (mg) (%) (mg) Cell line 1 0.3510.73 37 3.97 Cell line 2 0.32 9.54 25 2.37 Cell line 3 0.31 10.02 373.72 Cell line 4 0.42 10.01 40 4.00 Cell line 5 0.38 11.59 38 4.39 Cellline 6 0.43 11.10 43 4.75 Cell line 7 0.49 12.67 42 5.32 Cell line 80.33 8.96 22 2.01 Cell line 9 0.38 9.94 42 4.18 Cell line 10 0.39 10.9842 4.61

Purification and Characterization of IgGκλ Bispecific Antibody Expressedin CHO.

The supernatant of CHO cell pools transfected with bispecific andmonospecific constructs were used for purification. The monospecificanti-INFγ IgGλ, and the anti-IL6RC IgGκ were purified using Protein Aaffinity chromatography and desalted into PBS, whereas the bispecificIgGκλ antibodies were purified using the three step affinitychromatography process described in Example 8. The elution fractions andflow through of the different steps as well as the final purifiedsamples were analyzed by SDS-PAGE and IEF (FIGS. 16A-16B). Thespecificity of the IgGκλ was monitored after each purification step byELISA (FIGS. 17A-17D). The results demonstrate that the purificationprocess was robust and compatible with expression in CHO and yieldhighly pure IgGκλ bispecific antibody.

Example 12: Additional Examples of Bispecific IgGκλ Antibodies

Other examples of IgGκλ bispecific antibodies were isolated, expressedand purified as described in the Examples above. These included ananti-NusA/anti-INFγ IgGκλ bispecific antibody and ananti-IL6RC/anti-IL6RC IgGκλ bispecific antibody in which both combiningsites bind to IL6RC but carry a Kappa and a Lambda light chain. Thesepurified bispecific antibodies were analyzed by IEF along with theirrespective monospecific counterparts (FIG. 18). The results show thatthe pI of the bispecific antibody is always intermediate between the pIof the monospecific antibodies but that the differences can varysignificantly depending on sequence the light chain variable domain.This illustrates that purification of the bispecific antibody based oncharge differences might be difficult if the two light chains havesimilar biochemical properties and highlights the advantage of theaffinity purification approach of the invention.

Example 13: Generation of Bispecific IgGκλ Antibodies Using Two Kappa orTwo Lambda Variable Domains

Bispecific antibodies can also generated using two variable domains ofthe same type (Lambda or Kappa). As the affinity purification stepsrequire the presence of the constant Kappa and constant Lambda domainsof the light chains, any light chain variable domain can in principle befused to these constant domains to generate hybrid light chains asillustrated in FIGS. 3B-3C. This was demonstrated by using twoantibodies directed against INFγ and IL6RC isolated from fixed VHantibody libraries described in Example 1. In this case both antibodiesshare the same VH and have a Lambda light chain variable domain. TheVLambda domain of the anti-INFγ antibody was combined to the Lambdaconstant domain whereas the VLambda domain of the anti-IL6RC antibodywas combined with the Kappa constant domain. For the latter, threedifferent constructs were generated including different fusion pointsbetween the VLamda domains and the CKappa domain. In one construct(Hybrid 1) the fusion point was at the end of framework 4 (FR4) regionof the VLambda domain and include the whole the Constant Kappa domain(FIG. 19A). In another construct (Hybrid 2), the Lambda FR4 region wasreplaced by a Kappa FR4 region (FIG. 19B). In the third construct(Hybrid 3), the first 4 amino acids of the Constant Kappa domain weresubstituted by the 4 amino acids of the Constant Lambda domain (FIG.19C). These different hybrid Lambda-Kappa light chains were cloned alongwith the common heavy chain and Lambda light chain into the pNovi KMvector. Mammalian cell transfection, protein expression and three stepaffinity purification of the IgGκλ were performed as described in theExamples above. Analysis of the elution fractions indicated that theHybrid 3 light chain did not bind to the Kappaselect resin and thereforedid not allow for efficient bispecific antibody purification. The Hybrid1 and Hybrid 2 allowed for efficient IgGκλ bispecific purification.These purified IgGκλ bearing a hybrid light chain analyzed on an AgilentBionalyzer 2100 using a Protein 80 chip (Agilent Technologies) and thepeaks on the electropherogram corresponding to the light chains wereshown to be equivalent (FIG. 20). The results show that bispecificantibodies of the invention can be generated using two antibodies havinga common heavy chain and two light chains having Variable domains of thesame type (Kappa or Lambda).

Example 14: Both VH and VL of the Described Antibodies are Involved inAntigen Binding

In order to test the contribution of the common VH to antigen binding,different light chains of scFv bearing a common VH selected in Example 1and 4, were combined with two different VH that are different for theoriginal common VH. The different scFv could be expressed and purifiedas described in Example 3 and tested for binding against the targetagainst which they had been selected. Examples shown in FIGS. 21A-21Bshow that a scFv specific for INFγ and a scFv specific against IL6RC canonly bind to their respective targets when combined with the VH domainwith which they been originally selected and no binding is observed whencombined with two other VH domain that allow for their expression andpurification. The results indicate that despite the fact that diversityis restricted to the VL domain, both the common VH and the VL contributeto antigen binding.

Example 15: Development of ELISA for IgGκλ Bispecific AntibodyQuantification

A sandwich ELISA to quantify IgGκλ bispecific antibodies was developed.96-well Maxisorp (Nunc) plates were coated with 10 ug/ml mouse antihuman Lambda antibody (Southern Biotech) and incubated at 4° C.overnight. After washing (PBS Tween 20 at 0.05%, 3 washes), plate wasblocked with PBS-BSA 3% (Sigma) for 2 hours at room temperature.Purified IgGκλ standard was serially diluted in PBS-BSA 1% between 500ng/ml and 1 ng/ml to obtain a good linearity range for samplequantification. Depending on their origin the samples were diluted toenter the quantification range as follow: Crude CHO supernatant 1/1500;Protein A purified 1/15000. The samples were then diluted serially 1:2.After washing of the plates, 50 ul of each prepared dilution was addedin duplicate and incubated for 1 hour at room temperature. Plates werewashed again and incubated for 1 hour at room temperature with 50 ul of1:2000 anti human Kappa antibody (Southern Biotech). After the last wash(PBST 0.05%, 5 washes), the reaction was revealed with 50 ul of TMBsubstrate (Sigma) and stopped after 15 min by adding 50 ul of H2SO4(Sodium hydroxide 2N). The absorbance at 450 nm was recorded using aprecision microplate reader (Epoch, Witec). As the monospecificantibodies might affect the assay by binding to coated capture antibody,spiking experiments were performed by adding increasing amounts ofmonospecific IgGκ and monospecific IgGλ antibodies to the IgGκλbispecific standard. Different ratios were tested: (50% IgGλ bispecific,25% monospecific IgGκ, 25% monospecific IgGλ); (67% IgGκλ bispecific;33% monospecific IgGλ); (50% IgGλ bispecific, 50% monospecific IgGλ);(25% IgGκλ bispecific, 75% monospecific IgGλ); (50% IgGκλ bispecific,50% monospecific IgGκ).

The results shown in FIG. 22 indicate that the assay is not affectedsignificantly by monospecific antibodies and that therefore it can beused for IgGκλ bispecific antibody quantification in complex samplessuch cell culture supernatants. The ELISA was used to quantify IgGκλbispecific antibody in supernatant from stable CHO cell lines and aftertotal IgG purified from the same supernatants by Protein A affinitychromatography. The ELISA quantification results were compared to totalIgG content determined by Protein A HPLC or by absorption at 280 nm andare summarized in Table V. The concentrations obtained by ELISAcorresponded to 30-40% of total IgG, a proportion of bispecific that isexpected. The data shows that this assay can be used to determine theamount of IgGκλ bispecific antibody in a cell culture supernatant andfacilitates the screening of stable cell lines for their productivity inIgGκλ bispecific antibody.

TABLE V IgGκλ bispecific antibody quantification for different stableCHO cell lines using crude supernatants or total IgG by ELISA andcompared to ProteinA HPLC and A280 quantification results of total IgG.CHO supernatants Protein A purified Tot IgG Cell line Prot A HPLC ELISAA280 nm ELISA #11  0.3 mg/ml 0.18 mg/ml 4.38 mg/ml 2.3 mg/ml #14 0.42mg/ml 0.234 mg/ml  4.13 mg/ml 3.3 mg/ml #20 0.38 mg/ml 0.18 mg/ml 4.48mg/ml 1.7 mg/ml 10E4 0.39 mg/ml 0.19 mg/ml 4.81 mg/ml   3 mg/ml

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A method to generate an antibody mixturecomprising three or more monospecific antibodies and three or morebispecific antibodies, all having a common heavy chain, the methodcomprising: a. isolating an antibody or antibody fragment region havinga specificity determined by a heavy chain variable domain combined witha first light chain variable domain; b. isolating several antibodies orantibody fragments region having a different specificity determined bythe same heavy chain variable domain as the antibody of step a) combinedwith different light chain variable domains; c. co-expressing in a cell:i. a heavy chain polypeptide comprising the common heavy chain variabledomain fused to an immunoglobulin heavy chain constant region; ii. lightchain polypeptides comprising all the light chains of the antibodiesisolated in step a) and b) fused either to a light chain constant domainof the Kappa type or fused to a light chain constant domain of theLambda type; and d. recovering the heavy chain polypeptide and the lightchain polypeptides, thereby generating an antibody mixture comprisingthree or more monospecific antibodies and three or more bispecificantibodies, all having a common heavy chain.
 2. The method of claim 1further comprising the step of (d) purifying the antibody mixtureproduced in step c) from cell culture supernatant.
 3. The method ofclaim 2, wherein the purification step is performed using Kappa constantdomain specific, Lambda constant domain specific or both Kappa constantdomain specific and Lambda constant domain specific affinitychromatography media.
 4. The method of claim 1, in which the steps (a)and (b) are facilitated by the use of antibody libraries having a commonheavy chain and diversity confined to the light variable domain.
 5. Themethod of claim 4, wherein the antibody library is displayed on afilamentous bacteriophage, at the surface of yeast, bacteria ormammalian cells or used for ribosome or other type of in vitro display.